Antiviral compounds for high-risk, cancer-causing human papillomavirus (HPV) are extremely important even though anti-HPV vaccines are on the market. The proposed work determines the biophysics of interactions between antiviral polyamides (PAs) and their viral DNA targets, in order to determine the mechanism of action. The scope has been expanded from anti-HPV16 compounds to new anti-HPV compounds active against three cancer-causing viral subtypes. Active compounds eliminate viral episomes from human cell and tissue culture; they are potential drugs for the prevention of cervical cancer after HPV has been contracted. PAs having the same apparent DNA-binding preferences show dramatically different antiviral activities. It is proposed that a fundamental biophysical explanation of this behavior exists, one that goes beyond the known rules for PA-DNA interactions. Therefore, collaboration has begun around biophysical spectroscopy, DNA binding/footprinting assays, synthetic chemistry, virology and cellular-molecular biology. The aims are: Aim 1 Determine the binding position and affinity on the HPV16 genome of active PAs by footprinting the viral DNA as a function of PA concentration. The genome will be analyzed as overlapping 500 bp sequences using capillary electrophoresis and hydroxy radical footprinting. Binding constants determined for long DNA fragments will be compared with those from simpler models (minimal double-stranded or hairpin DNA). Independent physical methods such as fluorescence anisotropy (in competition binding studies), isothermal calorimetry and surface plasmon resonance will allow benchmarking against the literature and avoid artifacts. Aim 2 Footprint the HPV18 genome in vitro with antiviral PAs. Identify related binding sites for both HPV16 and 18. Carry out NMR structural work on DNA sequences of importance to both HPV16 and 18 as bound to active PAs; use NMR structural results as the basis for molecular modeling of additional interactions. Aim 3 Footprint active PAs on the HPV16 and 18 genomes in vivo in human cell culture. Examine the genomes using approximately 500 bp fragments generated by PCR that overlap to cover all sequence space. Determine experimental conditions and provide absolute binding constants with the aid of PA concentrations in cells measured by confocal and flow cytometry methods. Measure viral DNA concentrations vs. time using QPCR to make sure in vivo footprinting conditions are suitable. Identify structure-dependent uptake of PAs, potentially explaining why some isomeric PAs range from highly active to inactive antiviral agents. As an additional test of conclusions from footprinting, the site-directed mutagenesis of HPV18 sites identified during footprinting of antiviral PAs will help determine the importance of specific PA binding sites to the mechanism of antiviral action and viral episome maintenance. Provide new rules for interruption by polyamides of the high-risk HPV viral life cycle.